May 15, 2016 (Vol. 36, No. 10)

Overcoming Issues Associated with Antibody-Oligonucleotide Conjugation

First developed in 1992, immuno-PCR combines an ELISA (enzyme-linked immunosorbent assay) with PCR (polymerase chain reaction) to offer a significant increase in sensitivity compared with traditional ELISA. The technique works by coupling antibody specificity with DNA amplification.

However, despite demonstrating significantly greater sensitivity and a much wider dynamic range than an ELISA, immuno-PCR is not yet widely used.

The main reason for this is the time and complexity associated with conjugation of antibodies to oligonucleotides. Furthermore, the conjugation process can use considerable amounts of antibody, with losses being incurred during column-based purifications. New developments in antibody conjugation technology however mean that immuno-PCR can now be carried out much more efficiently.

Standard Immuno-PCR Process & Comparison with Standard ELISA

In the seminal immuno-PCR performed by Sano et al., Bovine Serum Albumin (BSA) was immobilized on microtiter plates, and was then detected with a monoclonal antibody to which a streptavidin-protein A chimera was attached. Biotinylated linear DNA was then added to the wells, amplified by PCR, and analyzed by agarose gel electrophoresis. This technique has subsequently evolved to incorporate rtPCR as a detection method, adding quantitation to the assay (Figure 1).

The main steps of an immuno-PCR assay are as follows:

1. Immobilization of antibodies specific for the protein target to the surface of a vessel
2. Washing to remove unbound antibody
3. Addition of sample
4. Washing to remove unbound sample
5. Addition of a second specific antibody, coupled to a DNA molecule
6. Washing to remove unbound antibody
7. DNA amplification and detection

In their 2005 publication, Lind and Kubista described the development of an immuno-PCR assay to quantify PSA in patient serum samples, and compared the results with those from a standard ELISA assay. PSA is a glycoprotein that is produced by the prostate gland and is a well-known marker for prostate cancer. PSA levels in the blood are usually extremely low. However, elevated serum concentrations may be indicative of prostate disease.

Lind and Kubista used two different monoclonal antibodies to PSA in their experiments, one for microplate coating, and the other for conjugation to a 67-base strand of linear DNA. In summary, preparation of the antibody-DNA conjugate involved activation of the antibody with thiol groups and activation of the DNA through the addition of an N-terminal amine group, column-based purification and elution of the activated antibody and DNA, mixing of the two solutions, column-based purification and elution of the conjugate, and removal of free antibody and DNA.

Figure 1. Schematic representation of an immuno-PCR assay. Capture antibody is immobilized on to the microplate surface and detected with an antibody-DNA conjugate. The DNA is then amplified and quantified by rtPCR.

Following production of the antibody-DNA conjugate, the immuno-PCR assay was developed and optimized. An overnight incubation was used to immobilize the capture antibody on to a microplate, after which the wells were washed and then blocked. During the blocking step, PSA standards or test serum samples were incubated with the antibody-DNA conjugate; these samples were then added to the microplate and incubated. After incubation the microplate wells were washed thoroughly. Two primers were used to generate a 65-base-pair-long product, which was fluorescently quantified by rtPCR.

Lind and Kubista then carried out an ELISA based on the same antibodies, to enable a comparison. Thirty serum samples were analyzed in both assay formats. The correlation between the two assays was found to be excellent, however the sensitivity of the immuno-PCR assay was found to be over 100 times higher than that of the ELISA (Figure 2).

Real-time immuno-PCR combines the exponential signal amplification of rtPCR with the specificity of an ELISA, providing greatly increased sensitivity and a significantly larger quantification range. Immuno-PCR can be used to detect extremely low levels of the antigen, often at 1,000-fold lower concentration than the levels which would be detected by an ELISA developed against the same target.

As well as enhanced sensitivity, further benefits of immuno-PCR are that it requires no additional equipment to that which is used for rtPCR; fewer incubation steps are required compared with ELISA, making it more robust and giving higher reproducibility; it is compatible with complex samples such as serum; and multiplexing is possible. Additionally, immuno-PCR is more cost-effective than many other assay formats.

Figure 2. Immuno-PCR (¦) and ELISA (?) readouts of standard samples, illustrating the superior sensitivity of immuno-PCR.

Improved Process

Although immuno-PCR offers many advantages over ELISA, the complexity issues associated with antibody-oligonucleotide conjugation have prevented the technique from being widely adopted. However, immuno-PCR can now be carried out much more efficiently as Innova Biosciences’ Thunder-Link PLUS technology enables efficient conjugation in just a few simple steps.

The conjugation process is unidirectional and allows only antibody-oligonucleotide complexes to be formed, with no antibody-antibody or oligonucleotide-oligonucleotide complexes produced. A positive control is also included in the kits to demonstrate successful conjugation, and an optional purification step enables any unconjugated oligonucleotides to be removed (Figure 3).

The simple Thunder-Link PLUS conjugation protocol involves separately adding the oligo and antibody to activation vials, and incubating both vials for 30 minutes at room temperature while washing the two desalt columns provided in the kit. The activated oligo mix is then added to the top of one column, and the activated antibody mix to the top of the other column. The liquid is pushed to the base of both columns, and eluted. The activated antibody and oligo are then added together, and incubated for one hour. The antibody-oligo conjugate is then purified with the Conjugate Clean Up Reagent provided.

Figure 3. The Thunder-Link PLUS conjugation process

Applications of Antibody-Oligonucleotide Conjugates

Antibody-oligonucleotide conjugates have huge potential for use in multiplexed protein diagnostic assays, since different reporter oligonucleotides can be conjugated to antibodies against specific protein targets. In addition to immuno-PCR, antibody-oligonucleotide conjugates are also applicable to other assay formats, including proximity ligation assays, proximity extension assays, and electrochemical proximity assays.

The next generation of tools in biomarker detection, antibody-oligo conjugates can now be generated easily and efficiently, hugely increasing accessibility across the scientific community.

1. Sano et al, Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA
Conjugates, Science 258 (5079) 120–122).
2. Lind et al, Development and evaluation of three real-time immuno-PCR assemblages for quantification of PSA, Journal of Immunological Methods 304 (2005) 107-116

Emma Easthope ([email protected]) is marketing executive at Innova Biosciences.

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